Accepted article

Rev. sci. tech. Off. int. Epiz., 2014, 33 (3), ... - ...
No. 22102014-00048-EN 1/18
The equine arteritis virus isolate from the
2010 Argentinian outbreak
This paper (No. 22102014-00048-EN) has been peer-reviewed, accepted, edited, and
corrected by authors. It has not yet been formatted for printing. It will be published in
December 2014 in issue 33-3 of the Scientific and Technical Review
G.E. Metz (1, 2), M.S. Serena (3), C.J. Panei (2, 3), E.O. Nosetto (1) &
M.G. Echeverría (1, 2)*
(1) Virology, Faculty of Veterinary Sciences, National University of
La Plata, 60 and 118, CC 296, 1900 La Plata, Argentina
(2) Members of CONICET-CCT (Consejo Nacional de
Investigaciones Científicas y Técnicas – Centro Científico
Tecnológico), La Plata, Argentina (Scientific Research Council)
(3) Immunology, Faculty of Veterinary Sciences, National University
of La Plata, 60 and 118, CC 296, 1900 La Plata, Argentina
*Corresponding author: [email protected];
[email protected]
Summary
A semen sample from a stallion infected during the 2010 equine
arteritis virus (EAV) outbreak was received for viral isolation prior to
castration of the animal. The virus was identified using a polyclonal
antibody immunofluorescence test. Reverse-transcription polymerase
chain reaction (RT-PCR) was used to amplify a region of the GP5
gene with primers GL105F and GL673R. The PCR products were
purified and sequences of both strands were determined in a
MegaBACETM 1000 with inner primers CR2 and EAV32. A
phylogenetic dataset was built with the previously reported sequences
of five strains isolated in Argentina, together with a group of selected
sequences obtained from GenBank. The unrooted neighbour-joining
tree was constructed using molecular evolutionary genetic analysis
(MEGA) and bootstrap analyses were conducted using 1,000 replicate
datasets. Evolutionary distances were computed using the maximum

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composite likelihood method. A NetNGlyc server analysis at the
Technical University of Denmark
(www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-
glycosylation in GP5 sequences. The phylogenetic analysis revealed
that the new strain (GLD-LP-ARG), together with other strains
previously isolated, belongs to the European group EU-1 but in a
different branch. The new strain shows 99% nucleotide identity with
strain A1 and 98.1% with the Belgian strain 08P178. Persistently
infected stallions and their cryopreserved semen constitute a reservoir
of EAV, which ensures its persistence in the horse population around
the world. These findings reinforce the importance of careful
monitoring of persistently infected stallions, as well as semen straws,
by RT-PCR or test mating, in accordance with national regulations.
Keywords
Argentina – Equine arteritis virus – Outbreak – Semen sample.
Introduction
Equine arteritis virus (EAV) was first isolated from fetal lung tissue
during an outbreak of respiratory disease and abortion in Bucyrus,
Ohio, in the United Stated of America (USA) (1). Until 2010, the
prevalence of EAV-infected stallions resident in Argentina was
thought to be very low. The first report of serological evidence was in
1984 (2), when a prevalence of 9.2% was found in the population of
warmblood horses sampled. The virus (strain LP01) was first isolated
in 2001 (3); later, strain LT-LP-ARG was isolated from the testicle of
a seropositive stallion that had been imported into Argentina in 1998
(4). In 1998, several EAV antibody-positive animals were detected in
two sport-horse breeding farms that practised artificial insemination
with imported semen. A follow-up study between July 2001 and
December 2003 found a prevalence of 45.8% in one of those farms;
three stallions were virus isolation-positive (strains LP02/R, LP02/C,
LP02/P) (5). Three new sequences of EAV from three archive semen
samples were obtained in 2008: one (RO-LP-ARG) from a stallion
housed on the breeding farm where the first strain of EAV was
isolated and the other two (RZ-LP-ARG, KB-LP-ARG) from the farm

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where the LP02 strains had been isolated (6). Before the 2010
occurrence of equine viral arteritis in Argentina, the virus had not
been involved in respiratory disease, abortion or foal death; the
prevalence was restricted to sport-horse breeds and to certain breed
lineages (7). Phylogenetic investigations of the virus have focused on
the hypervariable region of the GP5 gene, in both European and North
American strains (8, 9, 10). At present, Argentinian EAV strains all
belong to the same group (6, 11, 12). Although infrequently reported
in the past, confirmed outbreaks of arteritis appear to be on the
increase and in the past decade the disease has been reported in the
USA (13), France (14) and Belgium (15). Analysis of phylogenetic
relationships has been demonstrated to be an effective tool in tracing
the source of EAV infection.
In 2010, two mares on a thoroughbred breeding farm in Buenos Aires
Province seroconverted after insemination with frozen semen
imported from the Netherlands. The semen straws from the stallion
believed to be responsible for spreading the infection were submitted
to the Virology Laboratory, National Institute of Agricultural
Technology, Castelar, Buenos Aires, and EAV was isolated (16). Four
other farms that had used this semen were put into quarantine and all
the mares that had been inseminated with the infective semen were
found to be seropositive.
A high prevalence of EAV infection was also found at an equestrian
club located in central Buenos Aires, where two other mares had been
inseminated with infective semen from a stallion housed at the club.
Respiratory disease, fever, limb oedema and abortions were observed
during this outbreak. Twenty-seven jumping stallions seroconverted
and 22 persistently infected stallions were castrated. In the present
study, the ORF5 region of this EAV strain was analysed genetically
and compared with sequences of other strains from Argentina and
elsewhere.

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Materials and methods
Sample processing and virological methodology
The infected stallion at the equestrian club in central Buenos Aires
was a Hannoverian born in 2000 that had been housed at the club
since 2006; the horse was found to be seropositive in May 2010.
Before castration of the animal, a semen sample was processed for
routine virus isolation at the Virology Laboratory, Faculty of
Veterinary Sciences, National University of La Plata, as described
previously (3, 17). Briefly, serial tenfold dilutions (10–1 to 10–3) of the
sample were inoculated in duplicate onto RK13 cells grown in 6-well
plates. The cells were maintained in culture medium containing 2%
fetal bovine serum. Plates were incubated at 37°C in an atmosphere of
5% CO2 and examined daily for cytopathic effects. The virus was
identified using a polyclonal antibody immunofluorescence test, as
described previously (3).
Reverse-transcription polymerase chain reaction and
sequencing
Viral RNA was extracted from 500 µl of the supernatant of infected
RK13 cells with 500 µl of TRIzol® (Invitrogen) and precipitated with
isopropanol. Portions (5 µl) of RNA resuspended in distilled water
were used for cDNA synthesis using reverse transcriptase and random
hexamers (Promega). For PCR amplification, the primers GL105F and
GL673R, which flank a 546-nt region of the GP5 gene, were used
(18). Denaturation, annealing and extension consisted of 35 cycles at
94°C for 45 s, 60°C for 1 min and 72°C for 90 s, respectively. The
PCR products were run on 2% agarose gels, stained with ethidium
bromide, observed under UV light and purified using a PCR
purification kit. The sequences of both strands were determined in a
MegaBACETM 1000 with primers CR2 and EAV32, which flank a
519-nt region (8).

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Dataset and phylogenetic analysis
The phylogenetic dataset was built with five strains isolated in
Argentina, sequences reported previously (3, 4, 5, 6, 11), and a group
of selected sequences obtained from GenBank (Table I). Sequences
were edited using BioEdit software version 5 (19) and aligned in
molecular evolutionary genetic analysis (MEGA) software, version
4.0, using the ClustalW algorithm. Sequence pair distances were
calculated by DNAstar® (20). The unrooted neighbour-joining tree
was constructed using MEGA and bootstrap analyses were conducted
using 1,000 replicate datasets. Evolutionary distances were computed
using the maximum composite likelihood method (20).
Analysis of N-glycosylation sites
A NetNGlyc server analysis at the Technical University of Denmark
(www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-
glycosylation in GP5 sequences. This program predicts asparagines to
be N-glycosylated according to the Asn-Xaa-Ser/Thr sequons (where
Xaa is not Pro), with a threshold of 0.5.
Results
Viral isolation and reverse-transcription polymerase chain
reaction
After two passages in confluent monolayers of RK13 cells, cytopathic
effects were observed in cells inoculated with the seminal plasma at
10–1 and 10–2 dilutions when compared with control cells. The isolated
virus strain was named GLD-LP-ARG. After reverse transcription,
cDNA was obtained from the supernatants of the cultures. An
immunofluorescence test confirmed the presence of EAV antigen; no
virus-specific fluorescence was observed in control mock-infected
cells. Using primers specific for the GP5 gene, the cDNA gave a
visible 591-bp band in an ethidium bromide-stained agarose gel. No
bands were observed in the negative control used in the PCR. The
519-bp sequence was analysed and aligned with the Argentinian EAV